Our proposal explores the molecular pathogenesis and therapeutic targeting of diffuse large B-cell lymphomas (DLBCL), which are the most common form of non-Hodgkin's lymphoma. Since these tumors are markedly heterogeneous, we are interested in identifying mechanisms relevant to the broadest cross-section possible of DLBCL patients. A recent report suggested that heat shock protein 90 (Hsp90) is widely expressed in DLBCL. We independently confirmed and validated this result and showed that Hsp90 binds and protects from degradation the BCL6 transcriptional repressor and the NEMO subunit of the IKK complex, both of which are components of oncogenic pathways in DLBCL. Several groups have developed Hsp90 inhibitors. However, most of these molecules have a relatively narrow therapeutic window that makes it impossible to fully suppress tumor Hsp90 in vivo. The Chiosis laboratory developed a purine scaffold inhibitor of Hsp90 called PUH71. PUH71 more potently and specifically binds the fraction of Hsp90 that chaperones oncogenic client proteins within tumor cells vs. other Hsp90 inhibitors and readily kills DLBCL cells in vitro and in vivo. A single dose of PUH71 administered to mice remained in lymphomas for over 48 hours at therapeutic levels, but rapidly cleared from normal tissues in just a few hours. Accordingly, PUH71 exhibited a wider therapeutic window and thus greater anti-DLBCL efficacy than other Hsp90 inhibitors. Given these promising results, the National Cancer Institute Division of Cancer Treatment and Diagnosis is sponsoring the translation of PUH71 for use in clinical trials. Altogether, our preliminary mechanistic and pre-clinical data lead us to hypothesize that Hsp90 is a critical therapeutic target in a broad cross-section of DLBCLs and that DLBCLs become addicted to Hsp90 since it protects from degradation components of multiple pathways involved in the survival of DLBCL across its various subtypes. Given its superior pharmacologic properties, we predict that PUH71 will be a highly active in primary human DLBCL and will synergize with anti-lymphoma drugs that target complementary biological pathways. The current proposal will take advantage of the unique biochemical properties of PUH71 and use it as a bait to identify oncogenic Hsp90 client proteins in DLBCL in proteomics assays, coupled with functional validation studies. A second approach will explore the response of a spectrum of primary human DLBCLs to PUH71 and use expression profiling and bio-informatic tools to identify gene pathways that contribute to making DLBCLs biologically dependent on Hsp90. We will then use this information to design combinatorial-targeted therapy regimens for the treatment of DLBCL. This proposal will deliver mechanistic information concerning the functions of Hsp90 in lymphomas, and also will deliver new therapeutic regimens and biomarkers for testing in DLBCL patients.